Podcast
Questions and Answers
What are the functions of the -10 and -35 sequences in prokaryotic promoters?
What are the functions of the -10 and -35 sequences in prokaryotic promoters?
Which statement correctly describes the relationship between the template strand and the RNA transcript?
Which statement correctly describes the relationship between the template strand and the RNA transcript?
What determines the strength of a promoter in transcription initiation?
What determines the strength of a promoter in transcription initiation?
In what direction does RNA polymerase read the template strand during transcription?
In what direction does RNA polymerase read the template strand during transcription?
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What is a consensus sequence in the context of transcription?
What is a consensus sequence in the context of transcription?
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What is the typical sequence of the -10 region of the promoter recognized by the sigma factor?
What is the typical sequence of the -10 region of the promoter recognized by the sigma factor?
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Which of the following roles does the UP element play in transcription?
Which of the following roles does the UP element play in transcription?
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What role does the sigma factor play in the transcription process?
What role does the sigma factor play in the transcription process?
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Which of the following sequences is found in the -35 region of the promoter?
Which of the following sequences is found in the -35 region of the promoter?
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How does the strength of a promoter's match to the consensus sequence affect transcription levels?
How does the strength of a promoter's match to the consensus sequence affect transcription levels?
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Study Notes
PCR Outline
- Qualitative PCR only determines if a sequence is present
- Quantitative PCR determines if a sequence is present and the amount
- Regular/Traditional PCR is qualitative
- Advantages: fast, easy, sensitive, can amplify small amounts from degraded/fixed samples
- Disadvantages: often requires prior knowledge of the sequence, short products, infidelity of replication
- Components needed for a PCR reaction and their roles:
- Template DNA: blueprint for amplification
- Primers: dictate where to amplify
- DNA polymerase (Taq): synthesizes new strands, heat-resistant
- Nucleotides (dNTPs): building blocks of DNA
- PCR cycle steps and temperatures:
- Denaturation (95°C): separates DNA strands
- Annealing (~55°C): primers bind to target sequence
- Extension (72°C): new DNA synthesized
PCR Reaction
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Components needed for PCR reaction and their role:
- Template DNA: The DNA sequence to be copied
- Primers: Short DNA sequences that define the region to be amplified
- DNA Polymerase: An enzyme that synthesizes new DNA strands by adding nucleotides to the 3' end of the primer. Taq polymerase is commonly used due to its heat stability.
- Nucleotides (dNTPs): Deoxyribonucleoside triphosphates, the building blocks of DNA
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PCR cycle steps and temperatures:
- Denaturation: Heating the DNA to a high temperature (typically 94-98°C) to separate the double-stranded DNA into single strands.
- Annealing: Lowering the temperature (typically 50-65°C) to allow the primers to bind to their complementary sequences on the single-stranded DNA.
- Extension: Raising the temperature again (typically 72°C) to allow the DNA polymerase to synthesize new DNA strands by extending the primers.
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Why Taq Polymerase is used:
- Heat stable, can function at high temperatures required for PCR.
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Why PCR amplification is exponential
- Each DNA molecule produced can be used as a template for further replication.
Primer Design
- Length: Usually 20 nucleotides
- Avoid these when designing primers:
- Tandem repeats
- Secondary structures (hairpins)
- 3' end complementarity
Hot Start Methods
- Binding Protein Hot Start Method: Binding proteins bind to primers, then denaturation step inactivates them.
- Antibody Hot Start Method: Polymerase is bound by antibody preventing it from working until denaturation.
Genomic PCR vs. Reverse Transcriptase PCR (RT-PCR)
- Genomic PCR: Amplifies genomic DNA directly
- RT-PCR: Amplifies RNA by first converting it into cDNA
RT-PCR and cDNA
- RT-PCR converts RNA into cDNA using reverse transcriptase, then amplifies cDNA using PCR
- cDNA is single-stranded DNA produced from an RNA template using reverse transcriptase
qPCR
- Quantitative PCR
- Similar to regular PCR but measures the amplified DNA in real time
- Advantages: Monitor amplification real-time and accurate quantification of starting DNA material.
- Disadvantages: More expensive, requires more extensive instrumentation.
qPCR Data Analysis
- Ct value: Cycle number when fluorescence crosses a set threshold. Lower Ct values mean higher starting amounts of DNA target.
- Standard curve method: Use Ct to determine the starting DNA amount for unknown samples.
- AACt method: Used to compare Ct values of samples to a control (normalized)
Standard Curve
- Used to determine the initial starting amount of target template in experimental samples
- Slope measures reaction efficiency.
- Requires a dilution series of known template concentrations
Hot Start
- Method used to prevent non-specific amplification during PCR
- Keeps polymerase inactive until high temperatures are reached
Sequencing
- First Generation - Sanger Sequencing:
- Based on chain termination, low cost, high accuracy
- Second Generation (NGS) - NGS:
- High throughput, lower accuracy, cost-effective
- Third Generation - Long-Read Sequencing:
- Very long reads, relatively lower accuracy but cost-effective for particular applications
Emulsion PCR
- Amplifies DNA molecules in micro-droplets.
- Ensures each droplet contains only one DNA molecule, increasing specificity of reaction.
- Useful for high-throughput applications, minimizes contamination
Droplet Digital PCR (ddPCR™)
- More sensitive than qPCR
- Distributes template DNA into numerous droplets
- Measures DNA in individual droplets to determine concentration with high accuracy.
Sanger (dideoxy) Sequencing
- DNA polymerase synthesizes new DNA strands
- Chain terminator nucleotides (ddNTPs) create different lengths, fragments read on a gel.
Cycle Sequencing
- PCR-based sequencing method
- Similar to Sanger, but uses fluorescent ddNTPs instead of radioactive ones.
- Fragments are separated based on fluorescence signal.
Other Sequencing Methods
- Ion Torrent PGM: • Measures hydrogen ion released when nucleotides are added to the growing DNA strand.
- Molecular Beacons, TaqMan Probes: • Use fluorescent probes to detect DNA synthesis.
- Oxford Nanopore: • DNA strands pass through a nanopore, changes in current are detected to determine nucleotide sequence.
Assembly
- Combining short DNA fragments (reads) into longer, contiguous sequences (contigs) with overlapping regions
- Challenges: repetitive sequences, errors in sequencing, and the need to use reference genes.
Prokaryotic Transcription and Control Outline
- Transcription: Making mRNA from DNA
- Translation: Making protein from mRNA
- Operon: cluster of genes transcribed as a single mRNA in prokaryotes
- Promoter: DNA region where RNA polymerase binds to initiate transcription
- Leader Sequences: Region in some operons preceding the transcribed protein-encoding sequence, important in transcriptional regulation.
- Repressors: Proteins that bind to DNA sequences (operator) to block RNA polymerase, hindering transcription.
- Activators: Proteins that bind to DNA to stimulate RNA polymerase activity.
- Attenuation: a secondary regulatory mechanism of some operons, in which termination of transcript occurs early in some conditions.
- Constitutive: Always transcribed with or without a regulatory protein
- Inducible: Transcribed only when needed based on the environmental signals for an induced response
- Repressible: Transcribed unless blocked by a regulatory protein
RNA Types & Structures
- mRNA (messenger RNA): codes for proteins
- tRNA (transfer RNA): carries amino acids to ribosomes
- rRNA (ribosomal RNA): forms part of ribosomes
- ncRNA (non-coding RNA): various functions, many of which are regulatory (e.g., sRNA).
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Description
This quiz explores key concepts related to prokaryotic promoters, including the functions of -10 and -35 sequences, the relationship between template strands and RNA transcripts, and factors influencing promoter strength. Test your understanding of transcription initiation and consensus sequences in this engaging quiz.
